A "double-edged" role for type-5 metabotropic glutamate receptors in pain disclosed by light-sensitive drugs.

Knowing the site of drug action is important to optimize effectiveness and address any side effects. We used light-sensitive drugs to identify the brain region-specific role of mGlu5 metabotropic glutamate receptors in the control of pain. Optical activation of systemic JF-NP-26, a caged, normally inactive, negative allosteric modulator (NAM) of mGlu5 receptors, in cingulate, prelimbic and infralimbic cortices and thalamus inhibited neuropathic pain hypersensitivity. Systemic treatment of alloswitch-1, an intrinsically active mGlu5 receptor NAM, caused analgesia, and the effect was reversed by light-induced drug inactivation in in the prelimbic and infralimbic cortices, and thalamus. This demonstrates that mGlu5 receptor blockade in the medial prefrontal cortex and thalamus is both sufficient and necessary for the analgesic activity of mGlu5 receptor antagonists. Surprisingly, when light was delivered in the basolateral amygdala, local activation of systemic JF-NP-26 reduced pain thresholds, whereas inactivation of alloswitch-1 enhanced analgesia. Electrophysiological analysis showed that alloswitch-1 increased excitatory synaptic responses in prelimbic pyramidal neurons evoked by stimulation of BLA input, and decreased feedforward inhibition of amygdala output neurons by BLA. Both effects were reversed by optical silencing and reinstated by optical reactivation of alloswitch-1. These findings demonstrate for the first time that the action of mGlu5 receptors in the pain neuraxis is not homogenous, and suggest that blockade of mGlu5 receptors in the BLA may limit the overall analgesic activity of mGlu5 receptor antagonists. This could explain the suboptimal effect of mGlu5 NAMs on pain in human studies and validate photopharmacology as an important tool to determine ideal target sites for systemic drugs.

Up-regulation of the Trace Amine Receptor, TAAR-1, in the Prefrontal Cortex of Individuals Affected by Schizophrenia.

BACKGROUND AND HYPOTHESIS: Type-1 trace amine-associated receptors (TAAR1) modulate dopaminergic and glutamatergic neurotransmission and are targeted by novel antipsychotic drugs. We hypothesized that schizophrenia (SCZ) causes adaptive changes in TAAR1 expression in the prefrontal cortex. STUDY DESIGN: We measured TAAR1 mRNA and protein levels by quantitative PCR and immunoblotting in post-mortem prefrontal cortical samples obtained from 23 individuals affected by SCZ and 23 non-schizophrenic controls (CTRL). Data were correlated with a number of variables in both groups. STUDY RESULTS: TAAR1 mRNA levels were largely increased in the SCZ prefrontal cortex, and did not correlate with age, age at onset and duration of SCZ, or duration of antipsychotic treatment. For the analysis of TAAR1 protein levels, CTRL and SCZ were divided into 2 subgroups, distinguished by the extent of neuropathological burden. CTRL with low neuropathological burden (LNB) had lower TAAR1 protein levels than CTRL with high neuropathological burden (HNB), whereas no changes were found between LNB and HNB in the SCZ group. TAAR1 protein levels were lower in CTRL with LNB with respect to all SCZ samples or to SCZ samples with LNB. In the SCZ group, levels showed an inverse correlation with the duration of antipsychotic treatment and were higher in individuals treated with second-generation antipsychotics as compared with those treated with first-generation antipsychotics. CONCLUSIONS: The up-regulation of TAAR1 observed in the SCZ prefrontal cortex supports the development of TAAR1 agonists as new promising drugs in the treatment of SCZ.

mGlu3 Metabotropic Glutamate Receptors as a Target for the Treatment of Absence Epilepsy: Preclinical and Human Genetics Data.

BACKGROUND: Previous studies suggest that different metabotropic glutamate (mGlu) receptor subtypes are potential drug targets for treating absence epilepsy. However, no information is available on mGlu3 receptors. OBJECTIVE: To examine whether (i) changes of mGlu3 receptor expression/signaling are found in the somatosensory cortex and thalamus of WAG/Rij rats developing spontaneous absence seizures; (ii) selective activation of mGlu3 receptors with LY2794193 affects the number and duration of spikewave discharges (SWDs) in WAG/Rij rats; and (iii) a genetic variant of GRM3 (encoding the mGlu3 receptor) is associated with absence epilepsy. METHODS: Animals: immunoblot analysis of mGlu3 receptors, GAT-1, GLAST, and GLT-1; realtime PCR analysis of mGlu3 mRNA levels; assessment of mGlu3 receptor signaling; EEG analysis of SWDs; assessment of depressive-like behavior. Humans: search for GRM3 and GRM5 missense variants in 196 patients with absence epilepsy or other Idiopathic Generalized Epilepsy (IGE)/ Genetic Generalized Epilepsy (GGE) and 125,748 controls. RESULTS: mGlu3 protein levels and mGlu3-mediated inhibition of cAMP formation were reduced in the thalamus and somatosensory cortex of pre-symptomatic (25-27 days old) and symptomatic (6-7 months old) WAG/Rij rats compared to age-matched controls. Treatment with LY2794193 (1 or 10 mg/kg, i.p.) reduced absence seizures and depressive-like behavior in WAG/Rij rats. LY2794193 also enhanced GAT1, GLAST, and GLT-1 protein levels in the thalamus and somatosensory cortex. GRM3 and GRM5 gene variants did not differ between epileptic patients and controls. CONCLUSION: We suggest that mGlu3 receptors modulate the activity of the cortico-thalamo-cortical circuit underlying SWDs and that selective mGlu3 receptor agonists are promising candidate drugs for absence epilepsy treatment.

Synergic action of L-acetylcarnitine and L-methylfolate in Mouse Models of Stress-Related Disorders and Human iPSC-Derived Dopaminergic Neurons.

The epigenetic agents, L-acetylcarnitine (LAC) and L-methylfolate (MF) are putative candidates as add-on drugs in depression. We evaluated the effect of a combined treatment with LAC and MF in two different paradigms of chronic stress in mice and in human inducible pluripotent stem cells (hiPSCs) differentiated into dopaminergic neurons. Two groups of mice were exposed to chronic unpredictable stress (CUS) for 28 days or chronic restraint stress (CRS) for 21 day, and LAC (30 or 100 mg/kg) and/or MF (0.75 or 3 mg/kg) were administered i.p. once a day for 14 days, starting from the last week of stress. In both stress paradigms, LAC and MF acted synergistically in reducing the immobility time in the forced swim test and enhancing BDNF protein levels in the frontal cortex and hippocampus. In addition, LAC and MF acted synergistically in enhancing type-2 metabotropic glutamate receptor (mGlu2) protein levels in the hippocampus of mice exposed to CRS. Interestingly, CRS mice treated with MF showed an up-regulation of NFκB p65, which is a substrate for LAC-induced acetylation. We could also demonstrate a synergism between LAC and MF in cultured hiPSCs differentiated into dopamine neurons, by measuring dendrite length and number, and area of the cell soma after 3 days of drug exposure. These findings support the combined use of LAC and MF in the treatment of MDD and other stress-related disorders.

A Progressive Build-up of Perineuronal Nets in the Somatosensory Cortex Is Associated with the Development of Chronic Pain in Mice.

Chronic pain is sustained by a maladaptive form of neuronal plasticity occurring in all stations of the pain neuraxis, including cortical regions of the pain matrix. We report that chronic inflammatory pain induced by unilateral injection of complete Freund's adjuvant (CFA) in the hindpaw of male mice was associated with a progressive build-up of perineuronal nets (PNNs) in the contralateral somatosensory cortex (SSC), medial prefrontal cortex (mPFC), and reticular thalamic nucleus. In the SSC, the density of PNNs labeled by Wisteria floribunda agglutinin (WFA) was increased at both 3 and 7 d following CFA injection, but only after 7 d in the mPFC. The number of parvalbumin (PV)-positive interneurons enwrapped by WFA+/PNNs was also increased in all three brain regions of mice injected with CFA. Remarkably, PNN degradation induced by intracortical infusion of chondroitinase-ABC significantly reduced mechanical and thermal pain, and also reversed the increased frequency of IPSCs recorded in layer 5 pyramidal neurons of the contralateral SSC in CFA-injected mice. These findings suggest a possible relationship between cortical PNNs and nociceptive sensitization, and support the hypothesis that PNNs maintain their plasticity in the adult life and regulate cortical responses to sensory inputs.SIGNIFICANCE STATEMENT The brain extracellular matrix not only provides structural support, but also regulates synapse formation and function, and modulates neuronal excitability. We found that chronic inflammatory pain in mice enhances the density of perineuronal nets (PNNs) in the somatosensory cortex and medial prefrontal cortex. Remarkably, enzymatic degradation of PNNs in the somatosensory cortex caused analgesia and reversed alterations of inhibitory synaptic transmission associated with chronic pain. These findings disclose a novel mechanism of nociceptive sensitization and support a role for PNNs in mechanisms of neuronal plasticity in the adult brain.

Repeated episodes of transient reduction of oxygen exposure simulating aircraft cabin conditions enhance resilience to stress in mice.

Pilots and crew of domestic flights are exposed to transient periods of mild reductions of partial pressure of inspired oxygen each day, and this might have functional consequence on their performance in the long range. Here, we exposed mice to mild reductions of oxygen exposure (ROE) four times per day for 21 days by lowering oxygen partial pressure to levels corresponding to an altitude of about 2300 m, which is the quote of pressurization of the air cabin. Four groups of mice were studied: unstressed or stressed mice exposed to ROE or normoxic conditions. Mice were exposed to chronic unpredictable stress (CUS) for 28 days, and ROE was delivered in the last 21 days of CUS. In normoxic mice, CUS caused anhedonia in the sucrose preference test, anxiety-like behaviour in the open field test, learning impairment in the Morris water maze, reduced hippocampal neurogenesis, increased serum corticosterone levels and increased expression of depression-related genes (Pclo, Mthfr and Grm5) in the hippocampus. All these changes were reversed by ROE, which had little or no effect in unstressed mice. These findings suggest that ROE simulating air cabin conditions of domestic flights may enhance resilience to stress improving mood, anxiety and learning ability.

Genetic Deletion of mGlu3 Metabotropic Glutamate Receptors Amplifies Ischemic Brain Damage and Associated Neuroinflammation in Mice.

Backgroud: Type-3 metabotropic glutamate (mGlu3) receptors are found in both neurons and glial cells and regulate synaptic transmission, astrocyte function, and microglial reactivity. Here we show that the genetic deletion of mGlu3 receptors amplifies ischemic brain damage and associated neuroinflammation in adult mice. An increased infarct size was observed in mGlu3-/- mice of both CD1 and C57Black strains 24 h following a permanent occlusion of the middle cerebral artery (MCA) as compared to their respective wild-type (mGlu3+/+ mice) counterparts. Increases in the expression of selected pro-inflammatory genes including those encoding interleukin-1ß, type-2 cycloxygenase, tumor necrosis factor-α, CD86, and interleukin-6 were more prominent in the peri-infarct region of mGlu3-/- mice. In contrast, the expression of two genes associated with the anti-inflammatory phenotype of microglia (those encoding the mannose-1-phosphate receptor and the α-subunit of interleukin-4 receptor) and the gene encoding the neuroprotective factor, glial cell line-derived neurotrophic factor, was enhanced in the peri-infarct region of wild-type mice, but not mGlu3-/- mice, following MCA occlusion. In C57Black mice, the genetic deletion of mGlu3 receptors worsened the defect in the paw placement test as assessed in the contralateral forepaw at short times (4 h) following MCA occlusion. These findings suggest that mGlu3 receptors are protective against ischemic brain damage and support the way to the use of selective mGlu3 receptor agonists or positive allosteric modulators in experimental animal models of ischemic stroke.

Behavioural and biochemical responses to methamphetamine are differentially regulated by mGlu2 and mGlu3 metabotropic glutamate receptors in male mice.

Group II metabotropic glutamate receptors (mGlu2 and mGlu3 receptors) shape mechanisms of methamphetamine addiction, but the individual role played by the two subtypes is unclear. We measured methamphetamine-induced conditioned place preference (CPP) and motor responses to single or repeated injections of methamphetamine in wild-type, mGlu2-/-, and mGlu3-/-mice. Only mGlu3-/-mice showed methamphetamine preference in the CPP test. Motor response to the first methamphetamine injection was dramatically reduced in mGlu2-/-mice, unless these mice were treated with the mGlu5 receptor antagonist, MTEP. In contrast, methamphetamine-induced sensitization was increased in mGlu3-/-mice compared to wild-type mice. Only mGlu3-/-mice sensitized to methamphetamine showed increases in phospho-ERK1/2 levels in the nucleus accumbens (NAc) and free radical formation in the NAc and medial prefrontal cortex. These changes were not detected in mGlu2-/-mice. We also measured a series of biochemical parameters related to the mechanism of action of methamphetamine in naïve mice to disclose the nature of the differential behavioural responses of the three genotypes. We found a reduced expression and activity of dopamine transporter (DAT) and vesicular monoamine transporter-2 in the NAc and striatum of mGlu2-/-and mGlu3-/-mice, whereas expression of the DAT adaptor, syntaxin 1A, was selectively increased in the striatum of mGlu3-/-mice. Methamphetamine-stimulated dopamine release in striatal slices was largely reduced in mGlu2-/-, but not in mGlu3-/-, mice. These findings suggest that drugs that selectively enhance mGlu3 receptor activity or negatively modulate mGlu2 receptors might be beneficial in the treatment of methamphetamine addiction and associated brain damage.

Developmental up-regulation of NMDA receptors in the prefrontal cortex and hippocampus of mGlu5 receptor knock-out mice.

mGlu5 metabotropic glutamate receptors are highly expressed and functional in the early postnatal life, and are known to positively modulate NMDA receptor function. Here, we examined the expression of NMDA receptor subunits and interneuron-related genes in the prefrontal cortex and hippocampus of mGlu5-/- mice and wild-type littermates at three developmental time points (PND9, - 21, and - 75). We were surprised to find that expression of all NMDA receptor subunits was greatly enhanced in mGlu5-/- mice at PND21. In contrast, at PND9, expression of the GluN2B subunit was enhanced, whereas expression of GluN2A and GluN2D subunits was reduced in both regions. These modifications were transient and disappeared in the adult life (PND75). Changes in the transcripts of interneuron-related genes (encoding parvalbumin, somatostatin, vasoactive intestinal peptide, reelin, and the two isoforms of glutamate decarboxylase) were also observed in mGlu5-/- mice across postnatal development. For example, the transcript encoding parvalbumin was up-regulated in the prefrontal cortex of mGlu5-/- mice at PND9 and PND21, whereas it was significantly reduced at PND75. These findings suggest that in mGlu5-/- mice a transient overexpression of NMDA receptor subunits may compensate for the lack of the NMDA receptor partner, mGlu5. Interestingly, in mGlu5-/- mice the behavioral response to the NMDA channel blocker, MK-801, was significantly increased at PND21, and largely reduced at PND75. The impact of adaptive changes in the expression of NMDA receptor subunits should be taken into account when mGlu5-/- mice are used for developmental studies.

Perineuronal nets are under the control of type-5 metabotropic glutamate receptors in the developing somatosensory cortex.

mGlu5 metabotropic glutamate receptors are highly functional in the early postnatal life, and regulate developmental plasticity of parvalbumin-positive (PV+) interneurons in the cerebral cortex. PV+ cells are enwrapped by perineuronal nets (PNNs) at the closure of critical windows of cortical plasticity. Changes in PNNs have been associated with neurodevelopmental disorders. We found that the number of Wisteria Fluoribunda Agglutinin (WFA)+ PNNs and the density of WFA+/PV+ cells were largely increased in the somatosensory cortex of mGlu5-/- mice at PND16. An increased WFA+ PNN density was also observed after pharmacological blockade of mGlu5 receptors in the first two postnatal weeks. The number of WFA+ PNNs in mGlu5-/- mice was close to a plateau at PND16, whereas continued to increase in wild-type mice, and there was no difference between the two genotypes at PND21 and PND60. mGlu5-/- mice at PND16 showed increases in the transcripts of genes involved in PNN formation and a reduced expression and activity of type-9 matrix metalloproteinase in the somatosensory cortex suggesting that mGlu5 receptors control both PNN formation and degradation. Finally, unilateral whisker stimulation from PND9 to PND16 enhanced WFA+ PNN density in the contralateral somatosensory cortex only in mGlu5+/+ mice, whereas whisker trimming from PND9 to PND16 reduced WFA+ PNN density exclusively in mGlu5-/- mice, suggesting that mGlu5 receptors shape the PNN response to sensory experience. These findings disclose a novel undescribed mechanism of PNN regulation, and lay the groundwork for the study of mGlu5 receptors and PNNs in neurodevelopmental disorders.

Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors.

Polymorphic variants of the gene encoding for metabotropic glutamate receptor 3 (mGlu3) are linked to schizophrenia. Because abnormalities of cortical GABAergic interneurons lie at the core of the pathophysiology of schizophrenia, we examined whether mGlu3 receptors influence the developmental trajectory of cortical GABAergic transmission in the postnatal life. mGlu3-/- mice showed robust changes in the expression of interneuron-related genes in the prefrontal cortex (PFC), including large reductions in the expression of parvalbumin (PV) and the GluN1 subunit of NMDA receptors. The number of cortical cells enwrapped by perineuronal nets was increased in mGlu3-/- mice, suggesting that mGlu3 receptors shape the temporal window of plasticity of PV+ interneurons. Electrophysiological measurements of GABAA receptor-mediated responses revealed a more depolarized reversal potential of GABA currents in the somata of PFC pyramidal neurons in mGlu3-/- mice at postnatal d 9 associated with a reduced expression of the K+/Cl- symporter. Finally, adult mGlu3-/- mice showed lower power in electroencephalographic rhythms at 1-45 Hz in quiet wakefulness as compared with their wild-type counterparts. These findings suggest that mGlu3 receptors have a strong impact on the development of cortical GABAergic transmission and cortical neural synchronization mechanisms corroborating the concept that genetic variants of mGlu3 receptors may predispose to psychiatric disorders.-Imbriglio, T., Verhaeghe, R., Martinello, K., Pascarelli, M. T., Chece, G., Bucci, D., Notartomaso, S., Quattromani, M., Mascio, G., Scalabrì, F., Simeone, A., Maccari, S., Del Percio, C., Wieloch, T., Fucile, S., Babiloni, C., Battaglia, G., Limatola, C., Nicoletti, F., Cannella, M. Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors.

Screening for Niemann-Pick type C disease in neurodegenerative diseases.

Niemann Pick type C (NP-C) is an autosomal recessive neurovisceral lysosomal storage disorder caused by NPC1 and NPC2 gene mutations. We screened for NP-C 24 patients with Progressive Supranuclear Palsy and 10 with Multiple System Atrophy cerebellar type (MSA-C). Among PSP patients, no NPC1 or NPC2 gene variants were detected. One patient with MSA-C (10%) resulted to carry a pathogenic missense NPC1 gene mutation (p.C184Y) in heterozygous state. NPC1 genes variants might represent a risk or susceptibility factor in the development of α-synucleinopathies such as MSA. The common pattern of lysosomal dysfunction might explain the pathophysiological link between these disorders.

Acid sensing ion channel 2: A new potential player in the pathophysiology of multiple sclerosis.

Acid-sensing ion channels (ASICs) are proton-gated channels involved in multiple biological functions such as: pain modulation, mechanosensation, neurotransmission, and neurodegeneration. Earlier, we described the genetic association, within the Nuoro population, between Multiple Sclerosis (MS) and rs28936, located in ASIC2 3'UTR. Here we investigated the potential involvement of ASIC2 in MS inflammatory process. We induced experimental autoimmune encephalomyelitis (EAE) in wild-type (WT), knockout Asic1-/- and Asic2-/- mice and observed a significant reduction of clinical score in Asic1-/- mice and a significant reduction in the clinical score in Asic2-/- mice in a limited time window (i.e., at days 20-23 after immunization). Immunohistochemistry confirmed the reduction in adaptive immune cell infiltrates in the spinal cord of EAE Asic1-/- mice. Analysis of mechanical allodynia, showed a significant higher pain threshold in Asic2-/- mice under physiological conditions, before immunization, as compared to WT mice and Asic1-/- . A significant reduction in pain threshold was observed in all three strains of mice after immunization. More importantly, analysis of human autoptic brain tissue in MS and control samples showed an increase of ASIC2 mRNA in MS samples. Subsequently, in vitro luciferase reporter gene assays, showed that ASIC2 expression is under possible miRNA regulation, in a rs28936 allele-specific manner. Taken together, these findings suggest a potential role of ASIC2 in the pathophysiology of MS.

Alterations in the α2 δ ligand, thrombospondin-1, in a rat model of spontaneous absence epilepsy and in patients with idiopathic/genetic generalized epilepsies.

OBJECTIVES: Thrombospondins, which are known to interact with the α2 δ subunit of voltage-sensitive calcium channels to stimulate the formation of excitatory synapses, have recently been implicated in the process of epileptogenesis. No studies have been so far performed on thrombospondins in models of absence epilepsy. We examined whether expression of the gene encoding for thrombospondin-1 was altered in the brain of WAG/Rij rats, which model absence epilepsy in humans. In addition, we examined the frequency of genetic variants of THBS1 in a large cohort of children affected by idiopathic/genetic generalized epilepsies (IGE/GGEs). METHODS: We measured the transcripts of thrombospondin-1 and α2 δ subunit, and protein levels of α2 δ, Rab3A, and the vesicular glutamate transporter, VGLUT1, in the somatosensory cortex and ventrobasal thalamus of presymptomatic and symptomatic WAG/Rij rats and in two control strains by real-time polymerase chain reaction (PCR) and immunoblotting. We examined the genetic variants of THBS1 and CACNA2D1 in two independent cohorts of patients affected by IGE/GGE recruited through the Genetic Commission of the Italian League Against Epilepsy (LICE) and the EuroEPINOMICS-CoGIE Consortium. RESULTS: Thrombospondin-1 messenger RNA (mRNA) levels were largely reduced in the ventrobasal thalamus of both presymptomatic and symptomatic WAG/Rij rats, whereas levels in the somatosensory cortex were unchanged. VGLUT1 protein levels were also reduced in the ventrobasal thalamus of WAG/Rij rats. Genetic variants of THBS1 were significantly more frequent in patients affected by IGE/GGE than in nonepileptic controls, whereas the frequency of CACNA2D1 was unchanged. SIGNIFICANCE: These findings suggest that thrombospondin-1 may have a role in the pathogenesis of IGE/GGEs.

Analgesia induced by the epigenetic drug, L-acetylcarnitine, outlasts the end of treatment in mouse models of chronic inflammatory and neuropathic pain.

Background L-acetylcarnitine, a drug marketed for the treatment of chronic pain, causes analgesia by epigenetically up-regulating type-2 metabotropic glutamate (mGlu2) receptors in the spinal cord. Because the epigenetic mechanisms are typically long-lasting, we hypothesized that analgesia could outlast the duration of L-acetylcarnitine treatment in models of inflammatory and neuropathic pain. Results A seven-day treatment with L-acetylcarnitine (100 mg/kg, once a day, i.p.) produced an antiallodynic effect in the complete Freund adjuvant mouse model of chronic inflammatory pain. L-Acetylcarnitine-induced analgesia persisted for at least 14 days after drug withdrawal. In contrast, the analgesic effect of pregabalin, amitryptiline, ceftriaxone, and N-acetylcysteine disappeared seven days after drug withdrawal. L-acetylcarnitine treatment enhanced mGlu2/3 receptor protein levels in the dorsal region of the spinal cord. This effect also persisted for two weeks after drug withdrawal and was associated with increased levels of acetylated histone H3 bound to the Grm2 gene promoter in the dorsal root ganglia. A long-lasting analgesic effect of L-acetylcarnitine was also observed in mice subjected to chronic constriction injury of the sciatic nerve. In these animals, a 14-day treatment with pregabalin, amitryptiline, tramadol, or L-acetylcarnitine produced a significant antiallodynic effect, with pregabalin displaying the greatest efficacy. In mice treated with pregabalin, tramadol or L-acetylcarnitine the analgesic effect was still visible 15 days after the end of drug treatment. However, only in mice treated with L-acetylcarnitine analgesia persisted 37 days after drug withdrawal. This effect was associated with an increase in mGlu2/3 receptor protein levels in the dorsal horns of the spinal cord. Conclusions Our findings suggest that L-acetylcarnitine has the unique property to cause a long-lasting analgesic effect that might reduce relapses in patients suffering from chronic pain.